The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.     

First trimester isolated fetal nuchal lucency: significance and outcome

BACKGROUND: In this retrospective study the postoperative alteration of intraocular pressure after cataract extraction and posterior chamber lens implantation was analyzed statistically. The influence on intraocular pressure between different positions of the loops (sulcus versus bag) was compared. PATIENTS AND METHOD: The posterior chamber lens implantation was performed on 267 eyes with 129 sulcus implanted and 138 in-the-bag implanted intraocular lenses. The average age of the patients was 74.8 respectively 71.9 years, with an equal ratio of sex. In surgery the viscoelastic experimentation of the nucleus was done via corneoscleral cut. A x-crossed suture sealed the corneoscleral wound followed by subconjunctival injection of corticosteroids. The postoperative medication for 4-6 weeks consisted of a corticosteroid and antibiotic combination. RESULTS: The average intraocular pressure of the sulcus implanted respectively in the bag implanted intraocular lenses increased from 15.4 mm Hg respectively 15.9 mm Hg preoperatively to 16.3 respectively 15.8 mm Hg at the first postoperative day. After two months the intraocular pressure decreased to 14.6 respectively 14.1 mm Hg. That means a pre- to postoperative difference of -0.8 respectively -1.8 mm Hg. CONCLUSIONS: In summary both groups showed a slight decrease in average intraocular pressure after two months. In comparison to the sulcus implanted intraocular lens the decrease of intraocular pressure was more than 1 mm Hg in case of the in the bag implanted lens. This decrease was statistically significant but may be considered of less clinical interest.     

Issues in the design and interpretation of studies to evaluate the impact of community-based interventions

Increasingly, epidemiologists are faced with the need to evaluate the impact of an intervention that is delivered at the level of a community or cluster of individuals, rather than at the individual level. This has profound implications for the design and interpretation of a study to evaluate its impact. We start by discussing the issues arising in the extension of the randomized double-blind controlled trial methodology to the evaluation of interventions delivered to clusters of individuals, or to whole communities, where the unit of randomization is a cluster of individuals rather than an individual. We then consider alternative approaches to design, discuss their relative strengths and weaknesses and present a framework of design options. Finally we propose a pragmatic approach to evaluation design in this setting. We believe that the answer lies in the judicious selection of different design elements, combined in such a way that when the evidence from each is presented together, a clear picture of the impact of the intervention emerges. We illustrate this using an example from the recent literature.     

Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sect. D 52:93-104, 1996), which includes the site of PII modification, was not required for the binding of small-molecule effectors but was necessary for the interaction with all three receptors. Mutations altering residues of this loop or affecting the nearby B loop of PII, which line a cleft between monomers in the trimeric PII, affected the interactions with protein receptors and the binding of small-molecule ligands. Thus, our results support the predictions made from structural studies that the exposed loops of PII and cleft formed at their interface are the sites of regulatory interactions.     

Analysis of prognostic factors and patterns of failure in dogs with malignant oral tumors treated with megavoltage irradiation

OBJECTIVE: To determine quality and duration of progression-free survival (PFS) time in dogs with malignant oral tumors after definitive megavoltage irradiation, to analyze prognostic factors for PFS time and patterns of failure, and to analyze the influence of tumor recurrence and development of metastasis on survival. DESIGN: Prospective clinical trial. ANIMALS: 105 dogs with squamous cell carcinoma, fibrosarcoma, or malignant melanoma of the oral cavity without evidence of metastasis. PROCEDURE: Dogs were treated with 48 Gy over 4 weeks on an alternate-day schedule of 4 Gy/fraction. Multivariate analysis was done by use of Cox's regression model to determine significant prognostic factors and by use of a competing risk model to determine the differential effects of prognostic factors on type of, and time to, failure. In 8% of the dogs, severe acute radiation reactions in the final week of treatment resulted in treatment discontinuation. In 7.6% of the dogs, chronic radiation reactions, including bone necrosis and fistula formation, developed. RESULTS: Prognostic factors that independently affected PFS time were histologic type and tumor T stage. Histologic type significantly influenced pattern of failure, but not time to failure, whereas clinical stage significantly influenced time to failure, but not type of failure. CLINICAL IMPLICATIONS: Irradiation was a safe and effective treatment of malignant oral tumors. Because the local efficacy of radiation was influenced only by tumor size, early treatment of oral tumors should improve the prognosis. In dogs without tumor recurrence, systemic metastases, rather than regional metastases, limited long-term survival after radiation therapy.     

Investigation of the selenium status of aborted calves with cardiac failure and myocardial necrosis

Lesions of heart failure, specifically cardiac dilation or hypertrophy along with a nodular liver (chronic passive congestion) and ascites, have been found in 4-5% of aborted bovine fetuses. In this study, a group of 22 such fetuses was compared with groups of aborted fetuses without lesions of heart failure and with nonaborted fetuses obtained from a slaughterhouse. The fetuses were necropsied, tissues were taken for histopathology, and samples were collected for routine bacteriologic and virologic examinations. Liver and kidney tissue was saved for selenium analysis. Histopathologic examinations of myocardium of fetuses with cardiac failure revealed myocardial necrosis and mineralization in 7 fetuses, lymphocytic myocarditis in 5 fetuses, myocardial fibrosis in 5 fetuses, or no microscopic lesions in 5 fetuses. Mean liver selenium levels were 5.5 mumol/kg in the fetuses with heart lesions, 6.5 mumol/kg in the fetuses without heart lesions and 7.5 mumol/kg in fetuses from the slaughterhouse; these differences were statistically significant. The results suggest that selenium deficiency in bovine fetuses may cause myocardial necrosis and heart failure. This study also provides data on normal liver and kidney selenium levels in bovine fetuses from the analyses of 19 nonaborted fetuses.     

Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.     

Anti-CD3 epsilon F(ab')2 fragments inhibit T cell expansion in vivo during graft-versus-host disease or the primary immune response to nominal antigen

This study was undertaken to distinguish between several mechanisms responsible for graft-vs-host disease (GVHD) protection in anti-CD3epsilonF(ab')2 fragment (Fr)-treated recipients: TCR down-modulation, deletion, failure of expansion, or anergy induction. To quantify alloreactive T cell expansion and function, thoracic duct lymphocytes (TDL) were analyzed. Sixfold fewer donor TDL T cells were recoverable from anti-CD3epsilonF(ab')2 Fr as compared with irrelevant F(ab')2 Fr-treated recipients at the time of peak T cell expansion in vivo. Kinetic analysis revealed that donor T cell expansion was inhibited and not simply delayed by anti-CD3epsilonF(ab')2 Fr. Similar proportions of TDL T cells in irrelevant and anti-CD3epsilonF(ab')2 Fr were undergoing apoptosis. Although TCR modulation was observed, donor TDL T cells had intact anti-host alloresponses as compared with irrelevant F(ab')2 Fr-treated recipients. Because donor CD4+ T cells are primarily responsible for GVHD in this model, an adoptive transfer system was used in which the function and kinetics of expansion of OVA-specific CD4+ TCR transgenic cells could be physically tracked. Relevant Fr severely blunted CD4+ TCR transgenic T cell clonal expansion after OVA administration. Nonviable transgenic and nontransgenic T cells were proportionally similar in OVA-pulsed recipients, regardless of whether relevant or irrelevant F(ab')2 Fr were given. After discontinuing Fr, transgenic T cells were found to have intact in vitro OVA-specific responses. Our current and previous results suggest that reduced donor T cell expansion and T cell depletion both contribute to GVHD protection by anti-CD3epsilonF(ab')2 Fr. These data have implications for designing therapeutic approaches directed toward TCR targeting in humans.     

Balancing demands of cancer surveillance among survivors of thyroid cancer

As part of our investigation of structure-activity relationships in a Candida rugosa lipase-catalyzed ester hydrolysis reaction, methyl 2-(octylsulfinyl)benzoate has been studied, with respect to rate and enantioselectivity behaviour, using two different lipase preparations. A chiral normal-phase liquid chromatography method has been developed for determination of the experimental data, degree of conversion (c) and enantiomeric excess of the substrate (ees) or the product (eep), needed for calculation of the enantioselectivity in a kinetic resolution of this kind. A recently developed new class of network-polymeric chiral stationary phases, giving baseline-resolution with high selectivities for the ester substrates as well as their corresponding carboxylic acid products, permits, without any derivatization, an accurate and direct determination of the rate of the hydrolysis reaction and of the enantioselectivity from one and the same chromatogram.